P841: MOLECULAR MECHANISMS OF RESISTANCE TO IMMUNOMODULATORS IN MULTIPLE MYELOMA: AN IN VITRO STUDY
Authors: Raquel Alves, Ana Sofia Almeida, Maria Inês Costa, Beatriz Lapa, Joana Jorge, Ana Cristina Gonçalves, Catarina Geraldes, Ana Bela Sarmento-Ribeiro
Journal: HemaSphere (2023)

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: Multiple Myeloma (MM) is a hematologic neoplasm characterized by the presence of malignant monoclonal plasma cells in the bone marrow and the presence of myeloma-defining events. The introduction of Proteasome Inhibitors (PI), Immunomodulators (IMiDs) and Monoclonal Antibodies in MM treatment, had increased patients’ survival. Different IMiDs are currently used in clinical practice in combination with PI, such as Lenalidomide (LEN) and Pomalidomide (POM), having both cereblon as the main target. Nonetheless, this disease remains incurable, and resistance development is still a concern. Several mechanisms have been associated with resistance, including genetic alterations, glucose and drug efflux transporters dysregulation, alteration in molecular target, and dysregulation of signalling pathways of survival, oxidative stress and apoptosis. Aims: This project’s focus was to evaluate the molecular mechanism of resistance to IMiDs resistance, namely LEN and POM, in MM cells and assess the cross-resistance to Bortezomib (BTZ). Methods: LEN and POM resistant cell lines were developed from H929 and U-266 cells, both sensitive to these IMiDs, through continuous drug exposure (LEN-resistant cells: H929RL and U-266RL; POM-resistant cells: U-266RP). Resistant cells were maintained in presence of 100 μM of LEN or POM, respectively. Validation of resistant models and evaluation of cross-resistance to BTZ were performed by resazurin assay. Genomics alterations were evaluated using MLPA assay. Gene expression of ABC transporters (ABCB1, ABC2 and ABCG2), solute transporters (SLC2A1 and SLC2A11), cereblon (CRBN), NF-κB pathway (NFKB1 RELA, RELB, REL and IKBKB), KEAP1/NRF2 pathway (KEAP1 and NFE2L2), MYC, BCL2, and metabolizing-drug enzyme (NQO1) genes were analyzed by qPCR. Protein expression of P-gp, MRP1, BCRP, and NF-κB was evaluated by flow cytometry (FC). Activity of P-gp, MRP1/2, and BCRP was analyzed by FC. Levels of intracellular peroxide, peroxide anion, nitric oxide, mitochondrial reactive oxygen species and reduced glutathione were evaluated by fluorometry. The results were analyzed statistically (significance level of 95%). Results: The resistant models developed show different degrees of resistance according to IMiD and cell line. In LEN-resistant models, H929-RL and U-266RL cells presented an IC25 6.5x and over 1000x higher compared to parental cells, respectively. The U-266RP cells showed an IC25 500x higher than U-266 cells. In the resistant cells there was an increase in the percentage of cells expressing P-gp and MRP1 compared to sensitive lines. These models showed increased expression of SLC2A1, CRBN, RELA, RELB, KEAP1 and MYC, and decreased expression of REL, NFE2L2, BCL2 and NQO1. Regarding genomic alterations, the resistant lines had more amplifications and deletions compared with parental sensitive cells, and it was most noticeable in the LEN-resistant cell line U-266RL. Despite the resistance to IMiD, these showed sensitivity to BTZ, demonstrating that there is no cross-resistance between the studied IMIDs and BTZ. Summary/Conclusion In summary, our study shows that the increase in P-gp and MRP1 at the cell surface may be mechanisms related to the resistance to LEN and POM in MM. Furthermore, the IMiD resistance models developed did not show cross-resistance to BTZ, justifying the benefits of associating BTZ with IMIDs, even in a situation of resistance to these drugs. The remaining results should be further verified by more extensive molecular analysis Keywords: Immunomodulatory thalidomide analog, Multiple myeloma, Drug resistance